Abstract: Objective To investigate the expression of karyopherin α2 (KPNA2) and annexin A3 (ANXA3) in laryngeal cancer, and to explore their relationship with ferroptosis related genes (FRGs) and prognostic significance. Methods A total of 122 laryngeal cancer patients who were admitted to Nantong First People's Hospital Affiliated to Southeast University from January 2019 to January 2022 were selected and their tissue samples were collected. Quantitative PCR (qPCR) was used to detect the mRNA expression of KPNA2, ANXA3, and FRGs glutathione peroxidase 4 (GPX4), dipeptidyl peptidase 4 (DPP4), and solute carrier family 7 member 11 (SLC7A11) in laryngeal cancer and adjacent non-cancerous tissues. Immunohistochemistry (IHC) was employed to determine KPNA2 and ANXA3 protein expression in laryngeal cancer tissues. Protein expression characteristics of FRGs in laryngeal cancer were further analyzed using data from the CPTAC database with R software. Pearson correlation analysis was used to evaluate the relationships among KPNA2 mRNA, ANXA3 mRNA, and FRGs. Kaplan-Meier survival curves and Cox regression analyses were applied to identify independent prognostic factors for laryngeal cancer patients. Results Compared with adjacent non-cancerous tissues, the levels of KPNA2, ANXA3, DPP4, and SLC7A11 mRNA significantly increased, while GPX4 mRNA expression significantly decreased in laryngeal cancer tissues (P<0.05). KPNA2 mRNA and ANXA3 mRNA were positively correlated with DPP4 mRNA and SLC7A11 mRNA (r=0.718, 0.683; 0.686, 0.705; P<0.001) but negatively correlated with GPX4 mRNA (r=-0.632, -0.731; P<0.001). Moreover, KPNA2 mRNA and ANXA3 mRNA levels were positively correlated (r=0.725, P<0.001). The positive expression rates of KPNA2 and ANXA3 proteins in laryngeal cancer tissues were 60.66% (74/122) and 62.30% (76/122), respectively, which were significantly higher than those in adjacent tissues 6.56% (8/122) and 4.92% (6/122); P<0.001. Analysis of the CPTAC database revealed that DPP4 and SLC7A11 protein levels were elevated, while GPX4 protein levels were reduced in laryngeal cancer compared with normal laryngeal tissues (P<0.05). KPNA2 and ANXA3 protein expression was significantly associated with TNM stage and lymph node metastasis, with higher positivity rates in stage Ⅲ—Ⅳ and lymph node-positive cases (both P<0.05). Kaplan-Meier survival analysis demonstrated that the 3-year progression-free survival (PFS) rate was significantly lower in the KPNA2-positive group (45.95%, 34/74) than in the negative group (87.50%, 42/48), and similarly lower in the ANXA3-positive group (46.05%, 35/76) compared with the negative group (89.13%, 41/46) (both P=0.000). TNM stages Ⅲ—Ⅳ, lymph node metastasis, KPNA2 positivity, and ANXA3 positivity were independent prognostic risk factors for laryngeal cancer patients (all P<0.001). Conclusions KPNA2 and ANXA3 are highly expressed in laryngeal cancer tissues and closely correlated with FRGs. Both may serve as novel biological markers for evaluating the prognosis of patients with laryngeal cancer.